The success of gene therapy techniques depends largely on the ability to achieve a combination of stable chromosomal integration and high-level, regulated expression of transferred genes. Regulated gene expression is most easily achieved by means of large DNA fragments containing extensive cis-acting regulatory regions. For example, gene therapy for .beta.-globin disorders may require high-level, position-independent expression of extended gene and LCR sequences.
Many current techniques allow efficient transient transfection of cells in vitro and in vivo with large DNA fragments. However, subsequent chromosomal integration is very inefficient. To overcome low levels of integration, retroviral vectors which integrate very efficiently in permissive cells can be used. However, such vectors are greatly limited by constraints of size and sequence composition.
U.S. Pat. No. 4,959,317 (Sauer et al.) disclose the use of Cre-Lox site-specific recombination to achieve gene transfer in eukaryotic cells. However, the system described does not provide efficient or stable integration of transferred DNA into the host genome (see e.g., (Sauer et al. (1993) Methods in Enzymology 225: at 898). This is largely due to the fact that excision of transferred DNA out of the genome, by way of intramolecular exchange, predominates over integration of DNA into the genome, by way of intermolecular site-specific recombination.
A site-specific DNA recombination system which allows for highly efficient and stable integration of transgene sequences into genomic DNA would be greatly beneficial.